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The option 'Primer must span an exon-exon junction' will direct the program to return at least one primer (within a given primer pair) that spans an exon-exon junction. This is useful for limiting the amplification only to mRNA. You can also exclude such primers if you want to amplify mRNA as well as the corresponding genomic DNA. DESIGN PCR PRIMERS. BACKGROUND INFORMATION: For sites describing PCR theory, as well as companies marketing PCR products you might want to begin by visiting Highveld.For PCR techniques see PCRlink.com. There are several excellent sites for designing PCR primers: Primer3: WWW primer tool (University of Massachusetts Medical School, U.S.A.) – This site has a very powerful PCR primer. Genscript online pcr primer design tool for perfect PCR and sequencing primers design. Free gRNA design $199 /construct. GenScript Online PCR Primers Designs Tool Basic Standard Advanced. This online tool designs PCR primers for you. Sequence: Left primer Hybridization probe Right primer.
Primer Design Tool online, free
If you want to allow any of the unintended targets, check the box(es) next to the ones you accept and try again to re-search for specific primers Free Primer Design Tool For Sequencing
Primer-BLAST can only find non-specific primers for your template since there are other targets that are similar or identical to your template. You may choose to re-search for specific primers by allowing some of the highly similar off-targets if they are irrelevant to your PCR experiment. For example, not all transcript variants are expressed in all tissues, or you are not concerned about the predicted transcripts (i.e., XM_ accessions), or there are redundant database entries. Another example is, when you enter a raw sequence as PCR template rather than an accession, Primer-BLAST will likely report many off-targets. However, some of these might be your intended target that you should choose to allow. When Primer-BLAST does not need to avoid the highly similar off-targets in selecting unique primer regions, it typically has a much higher chance to find specific primers for your intended target.